Human papillomavirus E7 oncogenic protein is consistently expressed in HPV-expressing cervical cancers and is important in the induction and maintenance of cellular transformation. Thus, immunotherapy targeting E7 may provide an opportunity to treat HPV-associated cervical malignancies. We have previously shown that intradermal gene gun delivery of DNA vaccines encoding calreticulin (CRT) linked to E7 (CRT/E7) can significantly enhance E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. One major limitation of this DNA vaccination strategy is the limited transfection efficiency in vivo, which may limit the potency of DNA vaccines. Recent studies have described a novel approach to increase the transfection efficiency of the DNA vaccines in vivo using the femtosecond laser beam treatment. Thus, in the current proposal, we hypothesize that intradermal administration of the CRT/E7 DNA vaccine via needle injection or gene gun followed by femtosecond laser beam treatment would significantly enhance the transfection efficiency of the DNA vaccine, resulting in enhanced E7-specific T cell-mediated immune responses and antitumor effects in vivo. Specifically, we propose to: Specific Aim 1: Characterize the E7- specific CD8+ T cell immune responses and antitumor effects against an E7-expressing tumor using intradermal needle injection of the CRT/E7 DNA vaccine with or without laser treatment. Specific Aim 2: Characterize the E7-specific CD8+ T cell immune response and antitumor effects against an E7-expressing tumor using gene gun administration of the CRT/E7 DNA vaccine with or without laser treatment. Specific Aim 3: Perform a head-to-head comparison of intradermal needle injection of CRT/E7 DNA vaccine followed by laser treatment with gene gun administration of CRT/E7 DNA vaccine followed by laser treatment for their ability to generate E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. Specific Aim 4: Determine the mechanism of how laser treatment leads to enhancement in antigen- specific CD8+ T cell immune responses and antitumor effects in treated mice. The successful implementation of this study will represent an innovative approach for enhancing DNA vaccine potency. In addition, this study will potentially serve as an important foundation for future clinical translation. PUBLIC HEALTH RELEVANCE: Cervical cancer is the second leading cause of cancer deaths in women worldwide and human papillomavirus (HPV) has been identified as a necessary cause of cervical cancer. Our project aims to develop an innovative therapeutic HPV DNA vaccination strategy employing laser technology to improve DNA vaccine potency. Successful implementation of this study will represent an innovative approach for enhancing the potency of DNA vaccines targeting HPV for the treatment of cervical cancer.